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CBP manuscript 23538 – Part A 1
2
Different stressors induce differential responses of the CRH-stress system 3
in the gilthead sea bream (Sparus aurata) 4
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Juan A. Martos-Sitcha1,2*, Yvette S. Wunderink1,3, Justin Straatjes3, Arleta K. Skrzynska2, 6
Juan M. Mancera1 and Gonzalo Martínez-Rodríguez2 7
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(1) Department of Biology, Faculty of Marine and Environmental Sciences, University of 9
Cádiz, 11510, Puerto Real (Cádiz), Spain 10
(2) Department of Marine Biology and Aquaculture, Instituto de Ciencias Marinas de 11
Andalucía (CSIC), Apartado Oficial, 11510, Puerto Real (Cádiz), Spain 12
(3) Department of Organismal Animal Physiology, Institute for Water and Wetland Research, 13
Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135 6525 AJ Nijmegen, 14
The Netherlands 15
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*Corresponding author: 17
Dr. Juan Antonio Martos Sitcha 18
Department of Marine Biology and Aquaculture 19
Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC) 20
Apartado Oficial, 11510, Puerto Real (Cádiz), Spain 21
Tel.: +34 956 832612, Fax: +34 956834701 22
E-mail: [email protected] 23
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Abstract 25
The hypothalamus-pituitary-interrenal (HPI) axis, involved in the regulation of the 26
neuroendocrine stress responses, presents important players such as corticotropin-releasing 27
hormone (CRH, generally considered as the initiator of this pathway) and CRH-binding 28
protein (CRH-BP, considered as an antagonist of CRH function). CRH and CRH-BP 29
full-length cDNA sequences were obtained from Sparus aurata by screening a brain cDNA 30
library, and their phylogenetic analysis as well as their roles during acute and chronic stress 31
responses were assessed. mRNA expression levels and plasma cortisol concentrations were 32
measured by RT qPCR and ELISA, respectively, in S. aurata juveniles submitted to: i) 33
different environmental salinities in a short-time course response; and ii) food deprivation 34
during 21 days. In addition, osmoregulatory and metabolic parameters in plasma corroborated 35
a clear reorganization depending on the stress source/period. Salinity transfer induced stress 36
as indicated by enhanced plasma cortisol levels, as well as by up-regulated CRH and down-37
regulated CRH-BP expression values. On the other hand, food deprivation did not affect both 38
expression levels, although plasma cortisol concentrations were enhanced. These results 39
suggest that different stressors are handled through different stress pathways in S. aurata. 40
41
Keywords: 42
Cortisol, CRH, CRH-binding protein, environmental salinity, food deprivation, Sparus aurata 43
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3
1. Introduction 48
In teleost fishes, the hypothalamus-pituitary-interrenal (HPI) axis is stimulated under stress 49
situation. This axis starts with the production and release of corticotropin-releasing hormone 50
(CRH) from different hypothalamic nuclei, mainly the nucleus preopticus (NPO). CRH 51
stimulates the release of adrenocorticotropin hormone (ACTH), which is cleaved from the 52
precursor protein proopiomelanocortin (POMC), produced in adenohypophyseal corticotroph 53
cells. Subsequently, ACTH activates head kidney interrenal cells to produce and release the 54
typical stress hormone cortisol (Wendelaar Bonga, 1997; Flik et al., 2006; Bernier et al., 55
2009). 56
The mature form of CRH polypeptide consists of 41 amino acids, deriving from a larger 57
peptide of 160-210 amino acids, depending on the species, and signals via specific G-protein 58
coupled receptors of which two forms have been described: CRH-R1 and CRH-R2 (Vale et 59
al., 1981, Huising et al., 2008). CRH is highly conserved and can be found within virtually all 60
vertebrates, which indicates its endocrine importance. Besides CRH’s key function in the 61
stress response, this hormone is also involved in other processes, like feeding, digestion and 62
metabolism (Bernier et al., 2009; Yayou et al., 2011). In addition, studies in humans and other 63
mammals have also demonstrated that CRH plays a role in anxiety, arousal and depression 64
(Conti, 2012). 65
The biological activity of CRH can be regulated by a soluble binding protein, named 66
CRH-BP, since CRH presents a higher affinity for CRH-BP than for its own receptors 67
(Huising et al., 2004). Nevertheless, in mammals exist other ligands for CRH with different 68
affinities for the receptors and CRH-BP, like urocortin I (Ucn I), urocortin II (Ucn II) 69
/stresscopin-related peptide, and urocortin III (Ucn III), whereas fishes and amphibians 70
possess Urotensin I or sauvagine, respectively (Majzoub, 2006). 71
Like CRH, CRH-BP is mainly expressed in the NPO, and even co-locates with CRH, 72
suggesting a direct and rapid mechanism to regulate the stress response (Huising et al., 2004, 73
Flik et al., 2006). Additionally, physiological studies performed in teleostean species, indeed 74
have shown that CRH-BP can be considered as a strong modulator of the stress response 75
(Huising et al., 2004, Wunderink et al., 2011). 76
The degree of stress, or allostatic load, depends on the intensity and chronicity of the type of 77
stressor. Chronic exposure to stressors can lead to allostatic overload, which negatively 78
affects in reproduction, growth and immune function leading to diseases and reduced animal 79
welfare (Ellis et al., 2002, Conte, 2004, Ashley, 2007). Chronic stress is diagnosed by long-80
lasting, moderate changes of stress hormone levels as has been shown in several fish species 81
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(Rotllant et al., 2000, Wunderink et al., 2011, 2012). When a stressor is only exposed 82
shortly/intensively, a differential response is seen, defined by short duration, but more 83
pronounced alterations of stress hormone release (Rotllant and Tort, 1997, Ruane et al., 2002, 84
Huising et al., 2004, Doyon et al., 2005). In aquaculture, fish must cope with exposure to a 85
series of acute stressors such as transport weighing and handling, sorting/grading and sudden 86
environmental changes in, for instance, water temperature or salinity (Rotllant et al., 2001, 87
Arjona et al., 2007, Arjona et al., 2008, Mancera et al., 2008, Herrera et al., 2012), and might 88
become more susceptible when chronically stressed (Wunderink et al., 2011). To that account, 89
mapping the CRH-stress system contributes to a better understanding of the stress response 90
and may lead to improvement of aquaculture settings as well. 91
In gilthead sea bream (Sparus aurata) several studies have assessed changes in HPI axis due 92
to acute or chronic stress situations (Arends et al., 1999; Rotllant et al., 1997, 2000, 2001), but 93
no information exists on the role of CRH and CRH-BP during both stress situations. This 94
species is able to adapt to different environmental salinities adjusting their homeostasis in a 95
range of 5 to 60 ppt of salinity during 3 weeks (Laiz-Carrión et al., 2005; Sanguiao-Alvarellos 96
et al., 2005), being unable to withstand freshwater (Fuentes et al., 2010a). In part, this 97
plasticity is carried out by endocrine regulation, in which several hormones, including 98
cortisol, are involved (Takey and McCormick, 2013). However, a suddenly salinity transfer 99
can be considered as an acute stress situation for this species (Mancera et al., 1993; Laiz-100
Carrión et al., 2005). On the other hand, and related with feeding status of fish, long-term 101
adaptation to food deprivation has been proposed as a clear stress factor, where cortisol can 102
act as an important player in metabolic processes (Vijayan et al., 1993). Similarly, food 103
deprivation also enhanced plasma cortisol levels in S. aurata (Sangiao-Alvarellos et al., 104
2005b; Mancera et al., 2008). 105
In this study, the cDNAs coding for S. aurata CRH and CRH-BP peptides were cloned, 106
obtaining thus new molecular tools to study the neuroendocrine stress responses in this 107
species. Furthermore, the physiological roles of these genes in the acute and chronic stress 108
responses were characterized by monitoring their expression levels in S. aurata juveniles 109
submitted to: i) an acute stressor, viz. exposure to sudden environmental salinity changes, and 110
ii) a chronic stressor, viz. chronic exposure to food deprivation. 111
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2. Material and Methods 114
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2.1 Animals and experimental design 115
Juveniles of gilthead sea bream (Sparus aurata L., 213.13 ± 4.75 g body mass) were provided 116
by Planta de Cultivos Marinos (CASEM, University of Cádiz, Puerto Real, Cádiz, Spain; 117
Experimental animal facility registry numbers CA/4/CS and CA/3/U). Fish were fed a daily 118
ration of 1 % of their body weight with commercial pellets (Dibaq-Dibroteg S.A., Segovia, 119
Spain). All the experiments were performed with the Guidelines of the European Union 120
(2010/63/UE) and the Spanish legislation (RD 1201/2005 and law 32/2007) for the use of 121
laboratory animals. 122
123
2.1.1. Experimental design I: Short-term salinity transfer 124
Fish (n = 80, 192.11 ± 4.23 g body mass) were transferred to the wet laboratories at the 125
Faculty of Marine and Environmental Sciences (Puerto Real, Cádiz, Spain), where they were 126
acclimated for 14 days to sea water (SW, 38 ‰ salinity) in 400-L tanks in an open system 127
circuit (5.6 kg·m-3 density) under natural photoperiod (May, 2011) and constant temperature 128
(18-19 ºC). Afterwards, fish were directly transferred to one of the following environmental 129
salinities: SW (control group), low salinity water (LSW, 5 ‰ salinity, hypoosmotic transfer) 130
and high salinity water (HSW, 55 ‰ salinity, hyperosmotic transfer). These experimental 131
salinities were achieved by either mixing SW with dechlorinated tap water (LSW), or mixing 132
with natural marine salt (Salina de la Tapa, El Puerto de Santa María (Cádiz), Spain) (HSW). 133
Experimental groups were maintained in duplicate tanks (400-L volume each; n = 12 fish per 134
tank, 5.6 kg·m-3 initial density) under a closed recirculating water system. Water quality 135
criteria were checked at the end of the trial to confirm their stability during the 24 hours that 136
experiment lasted. On day 0 (10:00 AM), eight fish from the main tanks containing SW were 137
sampled (control time 0 before transfer). Then, on 4, 8, 12 and 24 hours after salinity transfer, 138
six fish from each experimental salinity (SW, LSW and HSW) were anaesthetized with a 139
lethal dose of 2-phenoxyethanol (1 mL·L-1 specific salinity water), weighted, heads separated 140
from trunks and sampled. 141
Blood samples were collected from the caudal peduncle into 1-mL ammonia-heparinised 142
syringes, and centrifuged (3 min at 10,000 g) to obtain plasma, snap-frozen in liquid nitrogen 143
afterwards and stored at -80 ºC until further analysis. Whole brains were put in a 1/10-relation 144
w/v of RNAlaterTM stabilization solution (Ambion®) for 24 hours at 4 ºC and then stored 145
at -20 ºC. No mortality was observed during the time that experiment lasted. Moreover, the 146
stocking density of each tank was restructured after each sampling point, by adjusting the 147
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final water volume in the tanks, to keep it constant throughout the experimental period and 148
between all tanks. 149
150
2.1.2. Experimental design II: Starving and re-feeding 151
Fish (n = 96; 235.31 ± 5.65 g body mass) were transferred to the wet laboratories at the 152
Faculty of Marine and Environmental Sciences (Puerto Real, Cádiz, Spain), where they 153
acclimated for 28 days to sea water (SW, 38 ‰ salinity) in five 1000-L tanks in an open 154
system circuit (4.3 kg·m-3 density) under natural photoperiod (March, 2011) and constant 155
temperature (18-19 ºC). After this acclimation period to SW, animals were maintained at the 156
following experimental conditions: 2 tanks fed with a daily ration of 1 % of their body mass 157
with commercial pellets (Dibaq-Dibroteg S.A., Segovia, Spain), and 3 tanks without receiving 158
food (n = 18 or 20 fish per tank). Furthermore, from day 14 after the start of the experiment, 159
fish from one tank maintained under food-deprived condition were fed again during 7 days 160
with a daily ration of 1 % of their body mass with the same commercial pellets described 161
above, constituting the re-feeding group. On day 0, eight fish from the main tanks containing 162
SW were sampled (control time 0 before transfer). Then, twelve fish from each experimental 163
group (control, starved and re-fed) on 7, 14 and 21 days after the start of the experiment, were 164
anaesthetized with a lethal dose of 2-phenoxyethanol (1 mL·L-1 specific salinity water), 165
weighted, heads separated from trunks and sampled. Blood samples and tissue biopsies were 166
taken as described above. No mortality was observed during the time that experiment lasted. 167
In addition, the stocking density of each tank was restructured as described above. 168
169
2.2. Plasma parameters 170
Plasma osmolality was measured with a vapor pressure osmometer (Fiske One-Ten 171
Osmometer, Fiske-VT, USA) and expressed as mOsm·kg-1. Glucose and lactate 172
concentrations were measured using commercial kits from Spinreact (Barcelona, Spain) 173
(Glucose-HK Ref. 1001200; Lactate Ref. 1001330) adapted to 96-well microplates. 174
Plasma cortisol levels were measured by enzyme-linked immunosorbent assay (ELISA) 175
adapted to microtiter plates as previously described for testosterone (Rodríguez et al., 2000). 176
Steroids were extracted from 5 µL plasma in 100 µL RB (PPB (Potassium Phosphate Buffer) 177
100 mM, NaN3 1.54 mM, NaCl 400 mM, EDTA 1 mM, BSA (Bovine Serum Albumin) 15 178
mM) and 1.2 mL methanol (Panreac), and evaporated during 48-72 hours at 37 ºC. Cortisol 179
EIA standard (Cat. #10005273), goat anti-mouse IgG monoclonal antibody (Cat. #400002), 180
specific cortisol express EIA monoclonal antibody (Cat. #400372) and specific cortisol 181
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express AChE tracer (Cat. #400370) were obtained from Cayman Chemical Company 182
(Michigan, USA). Standards and extracted plasma samples were run in duplicate. The 183
percentage of recovery was determined as 95 %, and evaluated as previously described in 184
others fish species (Barry et al., 1993; Mills et al., 2010). The inter- and intra-assay 185
coefficients of variation (calculated from the sample duplicates) were 3.20 ± 0.67 % and 6.41 186
± 0.73 %, respectively for salinity transfer, and 2.71 ± 1.03 % and 5.12 ± 0.48 %, respectively 187
for starving experiment. Cross-reactivity for specific antibody with intermediate products 188
involved in steroids synthesis was given by the supplier (cortexolone (1.6 %), 189
11-deoxycorticosterone (0.23 %), 17-hydroxyprogesterone (0.23 %), cortisol glucurinoide 190
(0.15 %), corticosterone (0.14 %), cortisone (0.13 %), androstenedione (<0.01 %), 191
17-hydroxypregnenolone (<0.01 %), testosterone (<0.01 %)). 192
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2.3. Cloning and sequencing 194
PCR was carried out on S. aurata brain cDNA with degenerate primers (Table 1) designed on 195
conserved regions of CRH-BP from Salmo salar (NM001173799), D. rerio (BC164122), 196
Haplochromis burtoni (GQ433718), Cyprinus carpio 1 (AJ490880), Cyprinus carpio 2 197
(AJ490881), and S. senegalensis (FR745428). For CRH, a specific probe obtained in Solea 198
senegalensis as previously described in Wunderink et al. (2011) was used. Both CRH and 199
CRH-BP probes were used for screening a brain cDNA library as described in Balmaceda-200
Aguilera et al. (2012). In vivo excision of 4 single positives of the screening were performed 201
using Escherichia coli XL-1-Blue MRF’ and SOLR strains (Stratagene, Agilent Technologies 202
Life Sciences). Excised pBluescript SK(-) containing the specific clone was double digested 203
by EcoRI and XhoI (Takara) and the products were revealed in a 1 % agarose gel stained with 204
GelRed™ (Biotium). Clones were fully sequenced in both strands by the dideoxy method 205
(Bioarray S.L., Alicante, Spain). 206
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2.4. Sequence analysis 208
Sequencing data were compiled, assembled and analyzed using nucleotide and protein 209
BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). eBiox (v1.5.1) software was used for 210
sequencing fragment assemblage, as well as for translation of the sequences to obtain the 211
open reading frames (ORFs). ClustalW2 software was used for protein alignment 212
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). Homology analysis of putative protein 213
sequences was run with NCBI blastp. 214
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2.5. Phylogenetic and evolutionary analyses 216
Phylogenetic analysis of the CRH-like and CRH-BP amino acid sequences was conducted 217
with MEGA5 software (Tamura et al., 2011) with the Neighbor-Joining algorithm (Saitou and 218
Nei, 1987) based on amino acid differences (p-distances) and pairwise deletion. Reliability of 219
the tree was assessed by bootstrapping (1000 replications). Amino acid sequences were 220
retrieved from the NCBI protein database (www.ncbi.nlm.nih.gov/pubmed), accessed in June 221
2014). 222
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2.6. RNA extraction and cDNA synthesis 224
Total RNA was extracted using the commercial kit NucleoSpin®RNA II kit (Macherey-225
Nagel) according to manufacturer’s instructions. Incubation with RNAse free DNase 226
(Macherey-Nagel) during 30 min at 37 ºC was used to eliminate potential genomic DNA 227
contamination. RNA concentrations were measured by spectophotometry and RNA quality 228
was assessed using the Agilent RNA 6000 Nano Assay Kits on an Agilent 2100 Bioanalyzer 229
(Agilent Technologies). Total RNA (500 ng) was reverse-transcribed in a 20 µL reaction 230
using the qScript™ cDNA synthesis kit (Quanta BioSciences). Briefly, the reaction was 231
performed using qScript Reaction Mix (1x final concentration) and qScript Reverse 232
Transcriptase (2.5 x final concentration). The reverse transcription program consisted in 5 min 233
at 22 °C, 30 min at 42° and 5 min at 85 °C. Only samples with a RNA Integrity Number 234
(RIN) higher than 8.5 were used for real time PCR. 235
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2.7. Real-time PCR (qPCR) 237
Specific primers for use in qPCR were designed by use of Primer 3 software (v. 0.4.0) 238
available at http://fokker.wi.mit.edu/primer3/input.htm in February 2011. Primer 239
oligonucleotide sequences are shown in Table 2. Previous to qPCR analysis, optimization of 240
qPCR conditions was made on primers annealing temperature (50 to 60 ºC), primers 241
concentration (100 nM, 200 nM and 400 nM) and template concentration (six 1:10 dilution 242
series from 10 ng to 100 fg of input RNA). Moreover, two negative controls, with i) RNA (10 243
ng/reaction) and ii) sterile water, were performed to detect possible gDNA contamination or 244
primer-dimers artefacts. The resulting curves had amplification efficiencies and r2 of 0.98 and 245
0.995 for CRH, 0.99 and 0.998 for CRH-BP, and 0.99 and 0,999 for β-actin, respectively. To 246
perform qPCR reactions, 4 µl cDNA (10 ng assumed from RNA input), specific forward and 247
reverse primers (200 nM each) and 5 µl PerfeCtaTM SYBR® Green FastmixTM (Quanta 248
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BioSciences) were used. qPCR (10 min at 95 °C; 40 cycles of denaturing for 15 s at 95 °C, 249
annealing and extension for 45 s at 60 °C; and a final melting curve from 60 °C to 95 °C for 250
20 min) was performed on a Mastercycler®epgradient S Realplex2 with Realplex software 251
(Eppendorf, version 2.2). The melting curve was used to ensure that a single product was 252
amplified by each primer pair. Results were normalized to S. aurata β-actin (acc. no. X89920) 253
owing its low variability (less than 0.5 CT) under our both experimental conditions. Relative 254
gene quantification was performed using the ∆∆CT method (Livak and Schmittgen, 2001). 255
256
2.8. Statistical analysis 257
Data were analysed by two-way ANOVA with salinity (LSW, SW, HSW) and time course 258
(day 0, 4, 8, 12 and 24 hours) as main factors for short-term salinity transfer, or by two-way 259
ANOVA with fed conditions (control and starving) and time course (days 0, 7, 14 and 21) as 260
main factors, and one-way ANOVA at day 21 for each treatment (control, starving and re-261
feeding) for starving experiment. These analysis were followed by a post-hoc comparison 262
made with the Tukey’s test, and using GraphPad Prism® (v.5.0b) software. Statistical 263
significance was accepted at P<0.05. Statistical parameters (P-value and F) obtained from 264
two-way ANOVA analysis in both sub-experiments are provided in Table 6. 265
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3. Results 268
3.1. Cloning and characteristics of S. aurata CRH and CRH-BP cDNA sequences 269
Complete sequences of sea bream CRH (GenBank acc. no. KC195964) and CRH-BP 270
(GenBank acc. no. KC195965) were obtained by screening a S. aurata brain cDNA library 271
using labelled probes. Sequencing revealed cDNAs to be 1,063 bp for CRH and 1,516 bp for 272
CRH-BP. 273
Figure 1 shows the obtained full-length nucleotide and deduced amino acid sequence of the 274
sea bream CRH peptide, which comprises an open reading frame (ORF) of 507 bp encoding a 275
169 amino acid protein whit 56-99 % similarity to other teleosts. ORF includes a conserved 276
signal peptide (M1 – A24), a cryptic motif (R55 – N66) and a mature peptide (S127 – F167), based 277
on alignment with other CRH sequences. Figure 2 shows a protein alignment done between 278
fish, amphibian, avian and mammalian CRH. The alignment shows 3 highly conserved 279
regions between these species, and scores between all the species are presented in Table 2A. 280
Moreover, as it has been observed in other species, the N-terminal dibasic cleavage site (R125 281
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– R126) of the mature peptide and the typical C-terminal amidation site (G168 – K169) are also 282
conserved. 283
On the other hand, the complete coding sequence of S. aurata CRH-BP is presented in Figure 284
3. cDNA sequence comprises an ORF of 969 bps encoding 323 amino acids with 58-90 % 285
sequence similarity to other teleosts, and included a signal peptide between amino acids M1 – 286
C26, the two conserved amino acids R59 and D65, and the ten conserved cysteine residues 287
(position numbers 63, 84, 107, 143, 186, 208, 239, 266, 279 and 320) involved in the 288
formation of five C-C disulphide loops. In addition, a protein alignment is shown in Figure 4 289
between fish, amphibian, avian and mammalian CRH-BP, revealing highly conserved 290
sequences at nucleotide (data not shown) and protein levels (Table 3B). 291
292
Phylogenetic analysis of non-mammalian and mammalian CRH-like and CRH-BP amino acid 293
sequences (Figure 5) indicated that S. aurata CRH clusters within the fish branch of CRH, 294
and just in the same branch of the CRH-family including CRH, UI, UcnI, UcnII and UcnII of 295
different species of fishes, amphibians, birds, and mammals. In addition, the vertebrate CRH 296
and UI/Ucn clusters together from the same clade, supported by a bootstrap value of 92. 297
Related to CRH-BP, vertebrates and invertebrates (insects) species are evolutionary more 298
distant, showing in vertebrates than amphibians, birds and mammals cluster independently 299
from fish species, supported by a bootstrap value of 100. 300
301
3.2. Effects of short-time salinity transfer (acute stress response) 302
Time courses of osmoregulatory and metabolic response of S. aurata to transfer to different 303
environmental salinities are shown in Table 4. These parameters did not show variations in 304
the control group (from SW to SW) along the time that experiment lasted. Plasma osmolality 305
revealed a clear time-course increased in its values in those fish submitted to hyperosmotic 306
transfer (from SW to HSW), showing a statistically increase (~12 %) on this parameter at the 307
end of the trial. In addition, a significant decrease (~12-15 %) was observed in osmolality 308
after hypoosmotic challenge (from SW to LSW) from 8 hours post-transfer compared with the 309
control group. On the other hand, fish transferred to HSW showed a significant increase of 310
around 55 to 65 % in plasma glucose, whereas in hypoosmotic transfer this enhancement was 311
of around 35 to 40 %. In addition, plasma lactate did not show variations in any of the 312
salinities tested in all experimental time. 313
Plasma cortisol levels rise in all groups tested, being significantly higher 4 hours after 314
hyperosmotic transfer, while for control group and hypoosmotic challenge (from SW to LSW) 315
11
a significant enhancement was not produce till 8 hours post-transfer. Later, in control group, it 316
returned to values from time 0 at 12 hours, remaining thus until the end of the experiment. At 317
24 hours post-transfer, cortisol levels dropped down to almost initial values in HSW group, 318
but not for fish transferred to LSW (Figure 6). 319
Expression levels of both CRH and CRH-BP after osmotic challenge are shown in Figure 7. 320
CRH mRNA expression presented similar time-course changes after LSW and HSW transfer. 321
Thus, both groups showed an increase of around 50 % in mRNA expression levels respect to 322
control group during all times tested, except at 8 hours where values close to the control 323
group were observed. Regarding CRH-BP mRNA expression, all groups showed a similar 324
pattern change at 4 hour post-transfer, increasing its values in a 50 %. After this time, control 325
group remained unchanged until the end of experimental time. CRH-BP mRNA expression 326
levels of fish submitted to hypoosmotic transfer showed a ~30 % increase in mRNA levels 327
compared to control group at 12 hours, while under hyperosmotic condition enhanced ~60 328
and ~35 % its expression at 12 and 24 hours post-transfer, respectively. 329
Statistical values of P-value and F obtained from the two-way ANOVA analysis for all 330
parameters tested in this sub-assay are shown in Table 5A. 331
332
3.3. Effects of starving and re-feeding situation (chronic stress response) 333
Time courses related to metabolic response of sea breams maintained under different feeding 334
conditions are shown in Table 5. Plasma glucose did not show changes in fish maintained 335
under normal fed conditions. Moreover, fish held under starving conditions significantly 336
enhanced its values respect to the control group, although the highest plasma glucose values 337
were observed in those fish re-fed during one week till day 21 (P-value: 0.041; F: 3.124). In 338
contrast, plasma lactate only showed statistically higher levels in fish maintained food-339
deprived during 21 days (P-value: 0.047; F: 2.963). 340
Plasma cortisol levels did not change in fish fed with a daily ration of 1 % of their body mass 341
and maintained as control group. However, fish submitted to starving situation significantly 342
increased these values around 7- to 8-fold respect to the control group during the first 14 days 343
of experiment, reaching the highest levels (11-fold) at the end of the trial (P-value: <0.001; F: 344
16.009). Moreover, re-feeding group during one week presented higher values respect to 345
fasting group, being 20-fold higher than the control group at the same sampling point (Figure 346
8). CRH mRNA expression was unchanged in all the groups and time points tested (Figure 347
9A). On the other hand, only the starved group showed around ~50 % of decreased values in 348
12
CRH-BP expression levels after 21 days of food deprivation respect to de control group and 349
the last time point (P-value: 0.031; F: 4.497) (Figure 9B). 350
Statistical values of P-value and F obtained from the two-way ANOVA analysis for all 351
parameters tested in this sub-assay are shown in Table 5B. 352
353
354
4. Discussion 355
In this study, the full-length cDNA sequences of CRH and CRH-BP in the teleost species S. 356
aurata was characterized, obtaining new tools to study their physiological roles in the acute 357
and chronic stress responses. 358
359
4.1. Sea bream CRH and CRH-BP sequences 360
The cDNA sequence of S. aurata CRH involves 1,073 bp that translates into a peptide of 169 361
amino acids. This is comparable in length with other teleost species like tilapia mossambica 362
(Oreochromis mossambicus) (167 amino acids), Senegalese sole (Solea senegalensis) (181 363
amino acids), zebrafish (Danio rerio) and common carp (Cyprinus carpio) (both 162 amino 364
acids) (van Enckevort et al., 2000, Huising et al., 2004; Wunderink et al., 2011). The CRH 365
prohormone can be subdivided into 3 regions: the signal peptide, the cryptic motive and the 366
mature peptide. S. aurata CRH prohormone appears to be between 42 % and 85 % identical to 367
other vertebrates. However, the mature peptide shows up to 68 % identity, which indicates 368
that the mature peptide is indeed the most important part of the hormone, namely the one 369
involved in receptor-binding. Likewise, S. aurata CRH-BP is highly conserved. CRH-BP is 370
known to be conserved throughout vertebrate and even invertebrate species (Huising and Flik, 371
2005), which underlines that CRH-BP might be as much as important in the stress response as 372
CRH to control all the processes in which it is involved in. In addition, both CRH and CRH-373
BP are strongly conserved throughout evolution. Both genes can be found in virtually all 374
vertebrates, and these genes can even be traced back as far as the insect linage. Furthermore, 375
Huising and Flik (2005) found CRH-BP sequence in Honeybee (Apis mellifera). This implies 376
that the origin dates back more than 400 million years (Knecht et al., 2011) and underlines the 377
importance of these genes, complemented by the structurally similar molecules involved. 378
379
4.2. Effects of salinity challenges 380
Hypoosmotic and hyperosmotic transfer induced changes in plasma osmolyte levels due to the 381
existing imbalance between the environmental and internal medium of the animal (Laiz-382
13
Carrión et al., 2005; Sangiao-Alvarellos et al. 2005a; Martos-Sitcha et al., 2013). Therefore, 383
during the adaptative period after salinity challenge of S. aurata specimens plasma osmolality 384
is disturbed, and an activation of several ion transporters located in different osmoregulatory 385
organs (mainly gills, intestine and kidney) is expected in order to maintain or adjust their 386
plasma osmolality within a certain range (Laiz-Carrión et al., 2005; Sangiao-Alvarellos et al. 387
2005a; Martos-Sitcha et al., 2013). In addition, our results related to plasma glucose suggest 388
the existence of an energetic reorganization that ensures the proper functioning of the 389
osmoregulatory system, although no variations in lactate values were presumable in a short-390
time response (24 hours) due to this metabolite has been described as one of the most 391
important metabolites during the chronic osmoregulatory period (Sangiao-Alvarellos et al., 392
2003, 2005a). 393
Moreover, this kind of acute stress agent activated HPI axis with early stimulation of CRH 394
and CRH-BP, followed by a plasma cortisol level enhancement as well as a metabolic and 395
osmoregulatory disorder. These data are in agreement with those obtained after acute stress 396
experiment performed on Cyprinus carpio (Huising et al., 2004), or even on S. aurata in 397
which this kind of stress can trigger an enhancement in cortisol values (Arends et al., 1999; 398
Sangiao-Alvarellos et al., 2005a). This hormone is also involved in other physiological 399
processes such as osmoregulation and metabolism (Wendelaar Bonga, 1997; Mommsen et al., 400
1999; McCormick, 2001), which explain the metabolic and osmoregulatory reorganization 401
observed. Fish in this experiment were maintained for 24 hours after transfer to both hypo- 402
and hyper-osmotic environment. Thus, plasma cortisol significantly increased during at least 403
the first 12 hours in both experimental transfers, indicating a primary stress response due to 404
salinity changes, similarly as previously observed after the same salinity transfer in this 405
species (Martos-Sitcha et al., 2013) and in Solea senegalensis (Herrera et al., 2012). In this 406
regard, plasma cortisol values as well as brain CRH and CRH-BP mRNA expression levels 407
showed a clear relationship in their values. Moreover, our results indicated a two-phase 408
activation of HPI axis with a good correspondence between plasma cortisol levels and CRH 409
and CRH-BP expression in the first moment after salinity transfer. Thus, just 4 hours post-410
transfer CRH and CRH-BP enhanced its mRNA levels, together with an increase in the 411
cortisol release into the bloodstream. However, at 8 hours post-transfer, the highest cortisol 412
values induced a clear negative feedback, which controls the down-regulation of both CRH 413
and CRH-BP factors. On the other hand, the subsequent decrease of plasma cortisol levels (12 414
hours post-transfer) is most likely the result of a drop in CRH expression combined with the 415
up-regulation of CRH-BP expression on the same sample-point in both extreme salinities. 416
14
Interestingly, at 24 hours (end point of experiment), fish submitted to hypoosmotic transfer 417
presented the highest plasma cortisol values, while that under hyperosmotic condition 418
returned to basal levels. This could reflect an osmoregulatory role for cortisol during 419
adaptative phase in S. aurata transferred to hypoosmotic environments, and it agrees with the 420
previously proposed hyperosmotic role for cortisol in this species increasing gill Na+,K+-421
ATPase activity, plasma osmolality, and ions after transfers from seawater to brackish water 422
(Mancera et al., 2002). Both groups showed up-regulation of CRH expression but only 423
hypoosmotic-transferred fish presented down-regulation of CRH-BP expression. These results 424
suggested that a coordination between both hypothalamic factors are thus clearly involved in 425
a fast regulation of plasma cortisol levels, inducing the strongly-pronounced, but short-lived, 426
cortisol response typical in acute stress situations (Huising et al., 2004). The high fluctuation 427
in CRH-BP expression compared to that in CRH expression might suggest that CRH-BP acts 428
stronger as a modulator of the acute stress response than CRH does, as it has been suggested 429
as well for the Senegalese sole (S. senegalensis) (Wunderink et al., 2011). Moreover, 430
activation of the hypothalamo-pituitary axis, with CRH as the first player implicated, and the 431
release of ACTH into the circulation by the pituitary is an integral part of the primary stress 432
response of fish (Donaldson, 1981; Sumpter et al., 1986; Balm and Pottinger, 1995). 433
Moreover, in the control group the lack of variations regarding with an expected increase in 434
CRH mRNA at 4 h in agreement with the cortisol enhancement at 8 h could suggest that only 435
handling stress required less amounts of stored protein (CRH), making that any additional 436
gene transcription initiated on top of the constitutive gene expression will remain 437
undetectable, although a contribution of daily rhythms on HPI-axis cannot be ruled out 438
(Montoya et al, 2010). 439
440
4.3. Effects of food deprivation 441
Studies assessing effects of food deprivation on stress axis in adult fish are scarce. Metabolic 442
reorganization after a prolonged stress source is expected due to the need to maintain vital 443
functions in the organisms. In fact, the reorganization observed in those fish maintained food 444
deprived is different compared with those submitted to an acute stress process (see above). 445
Thus, sea breams maintained under starvation revealed an enhancement in their plasma 446
glucose levels during the time that experiment lasted, together with a substantial increase in 447
lactate at the end of the trial. This fact demonstrated that i) food deprivation produced a 448
metabolic imbalance, and ii) re-feeding returned lactate concentration close to the control 449
values, but glucose remained enhanced as a consequence of high cortisol values (see below) 450
15
that probably produced higher glycogenolitic activity rates in several important metabolic 451
organs as liver, as has been previously demonstrated after different chronic stress situations, 452
including food deprivation (Sangiao-Alvarellos et al., 2003, 2005b). 453
Moreover, in this study food-deprived S. aurata enhanced plasma cortisol levels. Likewise, 454
elevated whole-body cortisol concentrations were found in zebrafish as a result of crowding 455
and food deprivation (Ramsay et al., 2006), and reduced stress resistance was demonstrated in 456
food-deprived Atlantic cod (Gadus morhua) (Olsen et al., 2008). Similarly, food-deprived S. 457
senegalensis juveniles significantly enhanced plasma cortisol levels (Costas et al., 2011a). In 458
addition, during early development, food-deprived S. senegalensis larvae showed an increase 459
in whole-body cortisol levels, as the result of an up-regulation of CRH expression and a 460
downregulation of CRH-BP expression (Wunderink et al., 2012). However, the lack of 461
variation in CRH mRNA expression as well as the down-regulation of CRH-BP values 462
suggests that, in S. aurata exposed to a long period of food deprivation, plasma cortisol level 463
could be regulated by both hypothalamic factors due to the putative lower regulation by the 464
soluble binding protein. Even so, specific changes in CRH-BP mRNA levels could varied in 465
each brain region depending on the stressor applied (Alderman et al., 2008), so a more 466
comprehensive study addressing i) each portion of the brain, deal with ii) different sources of 467
stress would be necessary to clarify the limited changes observed in our results. 468
In the re-fed group, fish showed the highest values of plasma cortisol and glucose together 469
with a lack of variation in CRH and CRH-BP mRNA expression. Although these results could 470
be a paradigm, the existence of such high values of cortisol could be explained by several 471
situations: i) the existence of a permanent state of alert to a situation of re-feeding after a 472
prolonged starving period (Uchida et al., 2003); ii) the stimulation of food intake by cortisol 473
(Bernier et al., 2004), where this hormone would act on food intake regulation enhancing the 474
stress recovery after food deprivation (Mommsen et al., 1999, Bernier et al., 2004); or iii) the 475
important role of cortisol during the metabolism reorganization (Mommsen et al., 1999). 476
Moreover, the absence of changes in CRH expression suggests that those processes focused in 477
cortisol production and release could be carried out through a different pathway. In fact, other 478
hormones and factors than just CRH and CRH-BP have been already described as putative 479
players involved in the stress response (Majzoub, 2006; Bernier et al., 2009), and the use of 480
CRH as a regulator of stress during food deprivation is somewhat of a paradox, since CRH 481
also acts as anorexigenic peptide (Uehara et al., 1998). Potential candidates to direct the stress 482
response independently of CRH are TRH through activating α-MSH (Lamers et al., 1991; 483
Rotlland et al., 2000; Van der Salm et al., 2004), and AVT nonapeptide that also stimulates 484
16
the release of ACTH (Baker et al., 1996). Thus, α-MSH is a key player in the neuroendocrine 485
stress response, depending on the type and source of the stressor (Wendelaar Bonga et al., 486
1995). Even so, the corticotrope activity of α-MSH is relatively weak (100 times less potent) 487
compared to that of ACTH (Wendelaar Bonga et al., 1995). Moreover AVT binding sites 488
have been described to be located in the zones occupied by corticotroph cells in 489
Dicentrarchus labrax (Moons et al., 1989) and Catostomus commersoni (Yulis and Lederis, 490
1987). In addition, the in vitro co-administration of AVT/AVP and CRF stimulate ACTH 491
secretion in preparations in vitro (Baker et al., 1996). Furthermore, AVT treatment plus hypo- 492
and hyperosmotic transfer enhanced plasma cortisol levels in S. aurata, suggesting a role of 493
AVT on stress axis activation in this species (Sangiao-Alvarellos et al., 2006). Recently, 494
Martos-Sitcha et al. (2013) demonstrated in S. aurata that pro-vasotocin mRNA synthesis and 495
pituitary storage of mature hormone is involved in the regulation of stress process after 496
salinity challenges, and also that food deprivation enhanced AVT storage in the pituitary 497
gland, suggesting that this hormone could acts as a paracrine factor on the ACTH cells (Gesto 498
et al., 2014). 499
500
501
5. Conclusions 502
Both, CRH and CRH-BP cDNA sequences were cloned in S. aurata. Their phylogenetic and 503
sequence analysis showed good gene conservation throughout evolution. Moreover, the 504
dynamics of change of osmoregulatory and metabolic parameters after two different sources 505
of stress (osmotic challenge –acute-, or food deprivation –chronic-) conditions confirmed the 506
internal derangement of the animals and its control mediated by the endocrine system. 507
Thus, the mRNA expression of these hormones, together with these changes reported on 508
plasma cortisol levels, indicated that the cortisol enhancement observed can be controlled by 509
different pathways, in which CRH seems to be regulated by CRH-BP during the acute stress 510
response, whereas during chronic stress (food deprivation) it could be controlled by other 511
factors acting as modulators (AVT or TRH hormones, among others). Even so, the 512
impossibility to discriminate variations in hypothalamic neurons alone could skew these 513
results in a complex endocrine system in which different pathways could regulate its proper 514
operation depending on the stressor. Moreover, the sequence in which stressors (acute or 515
chronic) occurs can produce different responses in this endocrine system as it has been 516
previously reported in S. senegalensis (Wunderink et al., 2011). 517
518
17
Acknowledgements 519
The authors wish to thank Planta de Cultivos Marinos (CASEM, University of Cádiz, Puerto 520
Real, Cádiz, Spain) for providing experimental fish. Experiment has been carried out at the 521
Campus de Excelencia Internacional del Mar (CEI-MAR) facilities from the University of 522
Cádiz. Study funded by project AGL2010-14876 from Ministerio de Ciencia e Innovación to 523
J.M.M. (Spain). J.A.M-S was supported by a PhD fellowship (FPU, Reference AP2008-524
01194) from Ministry of Education (Spain). 525
526
527
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Van der Salm AL, Pavlidis M, Flik G, Wendelaar Bonga SE (2004) Differential release of alpha-720
melanophore stimulating hormone isoforms by the pituitary gland of red porgy, Pagrus 721
pagrus. Gen Comp Endocrinol 135, 126-133. 722
van Enckevort FHJ, Pepels PPLM, Leunissen JAM, Martens GJM, Wendelaar Bonga SE, Balm PHM 723
(2000) Oreochromis mossambicus (tilapia) corticotropin-releasing hormone: cDNA sequence 724
and bioactivity. J Neuroendocrinol 12:177-186. 725
Vijayan MM, Maule AG, Schreck CB, Moon TW (1993) Hormonal control of hepatic glycogen 726
metabolism in food deprived, continuously swimming coho salmon (Oncorhynchus kisutch). 727
Canadian Journal of Fisheries and Aquatic Sciences 50:1676-1682. 728
Wendelaar Bonga SE (1997) The stress response in fish. Physiol Rev 77:591-625. 729
Wendelaar Bonga SE, Balm PHM, Lamers AE (1995) The involment of ACTH and MSH in the 730
stress-response in teleost fish. Neth J Zool 45:103-106. 731
23
Wunderink YS, Engels S, Halm S, Yúfera M, Martínez-Rodríguez G, Flik G, Klaren PHM, Mancera 732
JM (2011) Chronic and acute stress responses in Senegalese sole (Solea senegalensis): The 733
involvement of cortisol, CRH and CRH-BP. Gen Comp Endocrinol 171:203-210. 734
Wunderink YS, Martinez-Rodriguez G, Yufera M, Martin Montero I, Flik G, Mancera JM, Klaren PH 735
(2012) Food deprivation induces chronic stress and affects thyroid hormone metabolism in 736
Senegalese sole (Solea senegalensis) post-larvae. Comp Biochem Physiol A Mol Integr 737
Physiol 162:317-322. 738
Yayou K, Kitagawa S, Ito S, Kasuya E, Sutoh M (2011) Effect of oxytocin, prolactin-releasing 739
peptide, or corticotropin-releasing hormone on feeding behavior in steers. Gen Comp 740
Endocrinol 174:287-291. 741
Yulis CR, Lederis K (1987) Co-localization of the immunoreactivities of corticotropin-releasing factor 742
and arginine vasotocin in the brain and pituitary system of the teleost Catostomus 743
commersoni. Cell Tissue Res 247:267-273. 744
745
746
24
Tables 747
748
749 750 751 752 753 754 755 756 757
Table 1. Nucleotide sequences of degenerate primers designed for molecular identification of 758 CRH-BP partial cDNA sequence, and size amplified by each pair of primers. 759 760
761
762
763
764
765
766
767
768
769
770
771
772
773
Table 2. Nucleotide sequences of specific primers designed for qPCR analysis and size 774 amplified by each pair of primers. 775
776
Degenerate primers Nucleotide sequence Amplicon length
CRH-BP_Fw1 5’-CARTTYACMTTCACAGCAGA-3’ 718 bp
CRH-BP_Rv1 5’-CARGAGCTRCAGRYGATYAA-3’
CRH-BP_Fw2 5’-GTRTTYGAYTGGGTGATGAA-3’ 501 bp
CRH-BP_Rv2 5’-ATGAARRTYGGYTGTGAYAAC-3’
Primer Nucleotide sequence Amplicon length qCRH_Fw 5’-ATGGAGAGGGGAAGGAGGT-3’
176 bp qCRH_Rv 5’-ATCTTTGGCGGACTGGAAA-3’
qCRH-BP_Fw 5’-GCAGCTTCTCCATCATCTACC-3’ 147 bp
qCRH-BP_Rv 5’-ACGTGTCGATACCGCTTCC-3’
qb-actin_Fw 5’-TCTTCCAGCCATCCTTCCTCG-3’ 108 bp
qb-actin_Rv 5’-TGTTGGCATACAGGTCCTTACGG-3’
25
A) C
RH
S. aurata
S. senegalensis C
. carpio D
. rerio H
. sapiens M
. musculus
G. gallus
X. laevis
S. aurata 100
S. senegalensis
85 (78) 100
C. carpio
61 (78) 55 (60)
100
D. rerio
62 (78) 55 (63)
95 (97) 100
H. sapiens
49 (75) 44 (68)
59 (90) 53 (92)
100
M. m
usculus 46 (75)
41 (68) 50 (85)
51 (92) 79 (100)
100
G
. gallus 49 (75)
46 (68) 48 (90)
48 (92) 79 (100)
57 (100) 100
X
. laevis 42 (68)
45 (60) 47 (85)
48 (87) 53 (92)
50 (92) 57 (92)
100
777
B) C
RH
-BP
S. aurata S. senegalensis
C. carpio
D. rerio
H. sapiens
M. m
usculus G
. gallus X
. laevis S. aurata
100
S. senegalensis 60
100
C
. carpio 69
59 100
D
. rerio 69
61 97
100
H
. sapiens 57
53 62
63 100
M
. musculus
55 53
60 61
87 100
G. gallus
58 55
60 61
74 75
100
X. laevis
56 54
59 61
68 67
74 100
Table 3. A
) Alignm
ents scores of amino acid sequence identity for C
RH
(A) and C
RH
-BP (B
) sequences of various vertebrate species. For CR
H,
778 the identities w
ere given for the complete sequence and for the m
ature peptide (in parentheses). 779
780
781
26
Metabolite
Treatm
ent 0 hours
4 hours 8 hours
12 hours 24 hours
Osm
olality (m
Osm
·Kg-1)
SW →
LSW
SW →
SW
SW →
HSW
336.2 ± 12.5
a 317.3 ± 3.4
ab,* 340.5 ± 6.1
a 346.1 ± 5.4
ab
289.5 ± 2.4b,*
335.1 ± 3.9a
343.5 ± 10.3ab,#
295.3.6 ± 8.6b,*
336.2 ± 2.1a
353.2 ± 11.4ab,#
297.1 ± 6.4b,*
335.5 ± 3.2a
373.1 ± 5.6b,#
Glucose (m
M)
SW →
LSW
SW →
SW
SW →
HSW
8.143 ± 0.078
a 11.248 ± 0.244
b,* 8.122 ± 0.377
a 12.665 ± 1.054
b,*
11.406 ± 0.680b,*
8.445 ± 0.485a
13.639 ± 1.132b,*
11.693 ± 0.687b,*
8.319 ± 0.134a
13.626 ± 1.272b,*
11.111 ± 0.686b,*
8.325 ± 0.184a
13.402 ± 0.586b,*
Lactate (m
M)
SW →
LSW
SW →
SW
SW →
HSW
0.397 ± 0.016
a 0.416 ± 0.018
a 0.390 ± 0.026
a 0.396 ± 0.011
a
0.365 ± 0.009a
0.398 ± 0.040a
0.395 ± 0.027a
0.413 ± 0.032a
0.392 ± 0.027a
0.419 ± 0.035a
0.402 ± 0.029a
0.390 ± 0.011a
0.383 ± 0.031a
Table 4. Tim
e course changes in plasma osm
olality and metabolite (glucose and lactate) levels after transfer from
SW to different environm
ental 782
salinities (LSW, SW
and HSW
). Values are represented as m
ean ± S.E.M. (n = 7-8 fish per group). Significant differences betw
een sampling
783 points at the sam
e salinity are identified with different letters; different sym
bols show differences betw
een groups at the same tim
e (P<0.05, two-
784 w
ay AN
OV
A follow
ed by Tukey's test). 785
786
27
Metabolite
Treatm
ent D
ay 0 D
ay 7 D
ay 14 D
ay 21
Glucose (m
M)
Control
Starved Re-fed
4.844 ± 0.095a
4.838 ± 0.204a
5.297 ± 0.173b
4.872 ± 0.172a
5.351 ± 0.212b
4.759 ± 0.164a
5.374 ± 0.204b*
5.535 ± 0.276*
Lactate (m
M)
Control
Starved Re-fed
2.908 ± 0.199a
3.038 ± 0.154a
2.982 ± 0.183a
2.775 ± 0.110a
2.826 ± 0.224a
2.833 ± 0.261a
3.637 ± 0.467b*
2.589 ± 0.213a
Table 5. Tim
e course changes in plasma m
etabolite (glucose and lactate) levels in fish maintained under feeding, food deprivation and re-feeding
787 situations. V
alues are represented as mean ± S.E.M
. (n = 10-12 fish per group). Significant differences among sam
pling points at the same
788 condition are identified w
ith different letters; different symbols show
differences between groups at the sam
e time (P<0.05, one-w
ay AN
OV
A
789 follow
ed by Tukey’s test or Student t-test, in each case). 790
28
791
A Time Salinity Interaction Parameter P-value F P-value F P-value F
Osmolality 0.015 3.315 <0.001 66.670 <0.001 6.989 Glucose <0.001 13.309 <0.001 43.28 0.002 12.410 Lactate 0.871 0.309 0.940 0.061 0.971 0.278 Cortisol <0.001 32.131 <0.001 17.362 <0.001 8.544 CRH 0.005 5.660 0.001 7.487 0.594 0.812 CRH-BP <0.001 14.481 0.039 3.356 0.017 2.512
B Time Fed condition Interaction Parameter P-value F P-value F P-value F
Glucose 0.032 3.937 0.008 7.397 0.021 6.278 Lactate 0.002 4.988 0.032 3.937 0.465 0.872 Cortisol 0.002 5.222 <0.001 31.932 0.003 5.143 CRH 0.315 1.213 0.116 2.557 0.575 0.668 CRH-BP 0.011 4.233 0.743 0.109 0.724 0.443
Table 6. Statistical parameters (P-value and F) obtained from two-way ANOVA analysis in 792 fish transferred to different environmental salinities in a short-time response (A) or in fish 793 maintained under different feeding situations (B). 794
795
29
Legends to Figures 796
797
Figure 1. Nucleotide and deduced amino acid sequences of the sea bream (S. aurata) CRH 798
cDNA. The start and stop codon are presented in bold, underlined and italic. ORF is 799
highlighted in italic and underlined. The deduced amino acid sequence is displayed above the 800
nucleotide sequence. The predicted signal peptide M1-A24 and the conserved cryptic motif 801
R55-N66 are indicated in bold capitals. Predicted mature peptide S127-F167 is presented in bold 802
and underlined. The cleavage site and C-terminal amidation site are both underlined. 803
Accession number: KC195964. 804
805
Figure 2. Comparison of CRH amino acid sequences of four fish species [Sparus aurata 806
(AGO05917), Solea senegalensis (CBY78066), Cyprinus carpio (CAC84859) and Danio 807
rerio (ABS86029)], two of mammals [Homo sapiens (AAH11031) and Mus musculus 808
(AAI19037)], one of birds [Gallus gallus (CAF18561)] and one of amphibians [Xenopus 809
laevis (P49188)]. Alignment was carried out by ClustalW2 software (Larkin et al., 2007). 810
Gaps marked by hyphens have been inserted to optimize homology. Identical amino acid 811
residues are indicated in black. Signal peptide, cryptic motif and mature hormone structures 812
are noted behind the amino acid residues alignment. 813
814
Figure 3. Nucleotide and deduced amino acid sequences of the sea bream (S. aurata) 815
CRH-BP cDNA. The start and stop codon are presented in bold, underlined and italic. ORF is 816
marked in italic and underlined. The deduced amino acid sequence is displayed above the 817
nucleotide sequence. The predicted signal peptide M1-C26 is indicated in bold capitals. The 818
ten cysteines involved in the formation of five C-C disulfide bonds are boxed, underlined and 819
in bold. R59 and D65, probably implicated in ligand-binding with CRH are underlined and 820
indicated in bold capitals. Accession number: KC195965. 821
822
Figure 4. Comparison of CRH-BP amino acid sequences of four fish species [Sparus aurata 823
(AGO05918), Solea senegalensis (CBY78067), Cyprinus carpio (CAD35748) and Danio 824
rerio (NP_001003459)], two of mammals [Homo sapiens (NP_001873) and Mus musculus 825
(AAH61247)], one of birds [Gallus gallus (XP_003643006)] and one of amphibians [Xenopus 826
laevis (NP_001079273)]. Alignment was carried out by ClustalW2 software (Larkin et al., 827
2007). Gaps marked by hyphens have been inserted to optimize homology. Conserved 828
cysteine residues (essential for protein folding) are presented underlined, in bold, italics, and 829
30
highlighted in grey. Curved lines behind cysteine residues represent the formation of 830
disulphide bonds. R59 and D65, probably implicated in ligand-binding with CRH, are in italics 831
and double underlined. Identical amino acid residues are indicated in black. 832
833
Figure 5. Phylogenetic tree of CRH-like and CRH-BP amino acid sequences from several 834
fish species, including the sea bream (Sparus aurata), as well as amphibians, birds, mammals 835
and insects using Neighbor-Joining analysis and based on amino acid difference (p-distance). 836
Reliability of the tree was assessed by bootstrapping (1,000 replicates). GenBank and NCBI 837
Reference Sequences accession numbers are as follows: Sparus aurata CRH (AGO05917) 838
and CRH-BP (AGO05918); Oreochromis mossambicus CRH (CAB77056); Solea 839
senegalensis CRH (CBY78066) and CRH-BP (CBY78067); Danio rerio CRH (ABS86029), 840
UI (NP_001025351), UII (NP_998013) and CRH-BP (NP_001003459); Cyprinus carpio 841
CRH (CAC84859), UI (AAA49214) and CRH-BP (CAD35748); Oryzias latipes UI 842
(BAG16734), UcnII (BAG16730) and UcnIII (BAG16732); Platichthys flesus UI 843
(CAD56906) and UII (CAD56908); Takifugu rubripes CRH-BP (CAF18402); Salmo salar 844
CRH-BP (ACN11242); Osmerus mordax CRH-BP (ACO09096); Xenopus laevis CRH 845
(P49188), UcnI (NP_001086429), UcnIII (AAT70727), UII (NP_001267509) and CRH-BP 846
(NP_001079273); Spea hammondii CRH (AAP20883); Rana sylvatica CRH (AEQ37345); 847
Gallus gallus CRH (CAF18561); UcnIII (AGC65587), UII (NP_996873) and CRH-BP 848
(XP_003643006); Bos taurus CRH (AAI47873); Mus musculus CRH (AAI19037), UcnI 849
(NP_067265), UcnII (Q99ML8), Ucn III (Q924A4), UII (AAD55767) and CRH-BP 850
(AAH61247); Tupaia belangeri CRH (AFJ95881); Homo sapiens CRH (AAH11031), UcnI 851
(NP_003344), UcnII (Q96RP3), Ucn III (Q969E3), UII (AAD13070) and CRH-BP 852
(NP_001873); Rattus norvegicus UcnI (NP_062023), UcnII (Q91WW1), UII (EDL81198) 853
and CRH-BP (NP_631922); Ovis aries CRH-BP (NP_001009339); Apis mellifera CRH-BP 854
(NP_001012633); and Apis cerana cerana CRH-BP (ADG21869). 855
856
Figure 6. Plasma cortisol values in fish transferred from 38 ‰ to 38 ‰ (SW!SW), from 38 857
‰ to 55 ‰ (SW!HSW) or from 38 ‰ to 5 ‰ (SW!LSW). Values are represented as mean 858
± S.E.M. (n = 7-8 fish per group). Significant differences among sampling points at the same 859
salinity are identified with different letters; different symbols show differences between 860
groups at the same time (P<0.05, two-way ANOVA followed by Tukey’s test). 861
862
31
Figure 7. Expression levels of CRH (A) and CRH-BP (B) in fish transferred from 38 ‰ to 38 863
‰ (SW!SW), from 38 ‰ to 55 ‰ (SW!HSW) or from 38 ‰ to 5 ‰ (SW!LSW). Further 864
details as described in the legend of Figure 6. 865
866
Figure 8. Plasma cortisol values in fish maintained under feeding, food deprivation and re-867
feeding situations. Values are represented as mean ± S.E.M. (n = 10-12 fish per group). 868
Significant differences among sampling points at the same condition are identified with 869
different letters; different symbols show differences between groups at the same time 870
(P<0.05, one-way ANOVA or two-way ANOVA followed by Tukey’s test, in each case). 871
872
Figure 9. Hypothalamic expression levels of CRH (A) and CRH-BP (B) in fish maintained 873
under feeding, food deprivation and re-feeding situations. Values are represented as mean ± 874
S.E.M. (n = 6-7 fish per group). Further details as described in the legend of Figure 8. 875
876
32
Figure 1. Martos-Sitcha et al. 877
878
879
5’-atacttgtttctcctaagaagtgaaggagggcggcatctcgccaacta 48 ccttgcaaactgcacggctgttctggacctcctctaaagactgaagattcc 99 M K L N L L G T T V I L 12 tgctgatatcctgacatgaagctcaatttacttggcaccaccgtgattctg 150 L V A F L P R Y E C R A I E S P G 29 ctagttgccttcttaccccgctacgaatgtcgggctattgagagccctggc 201 G A L R V P A P Q T Q N S Q Q Q Q 46 ggtgccctgcgcgtcccagctccccaaacccaaaactcccagcagcagcaa 252 Q Q S G P I L E R L G E E Y F I R 63 cagcagtctggtcccatcctggagcggcttggagaggagtatttcatccga 303 L G N G D S N S F P S S S M Y P G 80 ctgggcaacggggactctaactctttcccatcttcgtccatgtatcccggc 354 G S P A I Y N R A L Q L Q L T R R 97 ggatcacctgcgatctacaacagagcgttgcaactccagctgacgcggcgt 405 L L Q G K V G N I R A L I S G F G 114 cttttacaaggaaaagttgggaacatcagggcgctcataagcggcttcgga 456 D R G D D S M E R G R R S E D P P 131 gaccgcggggacgactcgatggagaggggaaggaggtccgaggacccgccg 507 I S L D L T F H L L R E M M E M S 148 atttccctggatctgaccttccacctgctccgggagatgatggagatgtcc 558 R A E Q L A Q Q A Q N N R R M M E 165 agggcggaacagctggcccagcaagcgcaaaataacagaagaatgatggag 609 L F G K 169 ctcttcgggaaatgaagacctctttccagtccgccaaagatctccctttcc 660 tttcattttcttttcttcttcttctttttttttgttgcatttttaccatca 711 gcacaaaacatgctctgtacaatatagtgctgctttatcactctattattt 762 atagctttaacctcaaactatggagcttaaacgggcttgacttataatgat 813 ccgattgtaccttgccattttaatgttggtgtcaaatctgtagaattaagc 864 cgttcttcatgtttgagatgaaatactttgggttgacatgaaatactgcat 915 taacaaaactggcatactttgttttagatttcgaatcactgtatttatgat 966 atttatgtttgttaataaacttatgtgcaaccagtcattttctgttgtgca 1017 agagaacgtcttatatctatattttttaataaaaaaattaaaagcaaaaaa 1068 aaaaaaaaaaa -3’ 1079
33
Figure 2. Martos-Sitcha et al. 880
881
882
Figure 2. Martos-Sitcha et al. Signal peptide Sparus aurata MKLNLLGTTVILLVAFLPRYECRAIESPGGALRVPAPQTQNSQQQQQQ------------ 48 Solea senegalensis MKLNLFGTTVILLVAFLPRHECRAVDSRGGALRVLAPQTPNSQQQQQQQ------QQQQQ 54 Cyprinus carpio MKLNFLVTTVALLVAFPPPYECRAIESS-------SNQPAADPDGERQ------------ 41 Danio rerio MKLNFLVTTVALLVAFPPPYECRAIESS-------SNQPAADPDGERQ------------ 41 Homo sapiens MRLPLLVSAGVLLVALLPCPPCRALLSRGPVPGARQAPQHPQPLDFFQPPPQSEQPQQPQ 60 Mus musculus MRLRLLVSAGMLLVALSSCLPCRALLSRGSVP---RAPRAPQPLNFLQP----EQPQQPQ 53 Gallus gallus MKLQPLVCAGILLLALLPCHECRALSK---SPG--AARGALQQPDFFPQ---QQQQQQQQ 52 Xenopus laevis MKFQLWVSTGILLVSLLPCHECRAFIK---SPA--SSPGALLP-----------ALSNSQ 44 *:: : **::: . ***. .
Cryptic motif Sparus aurata SGPILERLGEEYFIRLGNGDSNSFPSSS-------------MYPGGSXAIYNRALQLQLT 95 Solea senegalensis SAPILERLGEEYFVRLGNEDSNSLPSSSSSS-------SSSMYPGGAPATYNRALQLQLT 107 Cyprinus carpio SPPVLARLGEEYFIRLGNRNQNSPRSPADS------------FPETS-QYSKRALQLQLT 88 Danio rerio SPPVLARLGEEYFIRLGNRNPTSPRSPADS------------FPETS-QYPKRALQLQLT 88 Homo sapiens ARPVLLRMGEEYFLRLGNLNKSPAAPLSPASSLLAGGSGSRPSPEQATANFFRVLLQQLL 120 Mus musculus --PVLIRMGEEYFLRLGNLNRSPAARLSPNSTPLTAGRGSRPSHDQAAANFFRVLLQQLQ 111 Gallus gallus TLPVLLRMGEEYFLRLGHLTKRPAGPFSASS-----GGHLRP---EASAELLRAAAAQLQ 104 Xenopus laevis --PFLLRMGEEYFLRLGNLHKHSPGSFPEAS----------------AGNFVRAVQQLQA 86 *.* *:*****:***: . . *.
Mature hormone Sparus aurata RRLLQGKVGNIRALISGFGDRG--DDSMERGRRSEDPPISLDLTFHLLREMMEMSRAEQL 153 Solea senegalensis RRLLQGKVGNIRALFSGFDDRG--DESMERGRRSEDPAISLDLTFHLLRGMMEMSRAEQL 165 Cyprinus carpio QRLLEGKVGNIGRLDGNYALRA--LDSVERERRSEEAPISLDLTFHLLREVLEMARAEQM 146 Danio rerio QRLLEGKVGNIGRLDGSYALRA--LDSMERERRSEEPPISLDLTFHLLREVLEMARAEQM 146 Homo sapiens LPRRSLDSPAALAERGARNALGGHQEAPERERRSEEPPISLDLTFHLLREVLEMARAEQL 180 Mus musculus MPQRSLDSRAEPAERGAEDALGGHQGALERERRSEEPPISLDLTFHLLREVLEMARAEQL 171 Gallus gallus G------SGSPEGDEGAG-------EAVEREKRSEEPPISLDLTFHLLREVLEMARAEQL 151 Xenopus laevis QQWSSQPGMRAASLDGADSPYSAQEDPTEKAKRAEEPPISLDLTFHLLREVLEMARAEQI 146 . . *: :*:*:..*********** ::**:****: Sparus aurata AQQAQNNRRMMELFGK 169 Solea senegalensis AEQAKNNEILMERYGK 181 Cyprinus carpio AQQAHSNRKMMEIFGK 162 Danio rerio AQQAHSNRKMMEIFGK 162 Homo sapiens AQQAHSNRKLMEIIGK 196 Mus musculus AQQAHSNRKLMEIIGK 187 Gallus gallus AQQAHSNRKLMEIIGK 167 Xenopus laevis AQQAHSNRKLMDIIGK 162 *:**:.*. :*: **
Figure 3. Martos-Sitcha et al.
!!
Figure 6. Martos-Sitcha et al.
M R V M E R T F R E Q L 12 5’-ctgcagacagagatgcgcgtgatggagcgcacgttccgcgagcagctg 48 F F L L L C A S V L K G D C R Y I 29 ttcttcctgctgttgtgcgcgtcggtgctgaagggagactgcaggtacatc 99 E N N E I S K D E L Y S F F N S E 46 gagaacaacgagatctccaaagatgagttatattctttcttcaactcggag 150 L K R E T T E E L M Y R R P L R C 63 ctgaagagagaaacaacggaggagttaatgtaccgtcgacctctacgctgt 201 L D M V A V E G Q F T F T A E R P 80 ctggacatggtggctgtggagggtcagttcaccttcacggccgagcgtcct 252 Q L S C A A F F M A E P N E V I T 97 cagctcagctgcgccgctttcttcatggccgagcccaacgaggtgatcacg 303 V E Y D N V D I D C R G G D F I T 114 gtggagtacgacaacgtcgacatcgactgcaggggaggagacttcatcacg 354 V F D G W V M K G E K F P S S Q D 131 gtgtttgacggctgggtgatgaaaggagagaagttccccagctcccaggat 405 H P L P L Y E R Y V D Y C D S G A 148 cacccgctgcctctgtacgagcgatatgtggattactgcgactcgggagcg 456 L R R S V R S S Q N V A M I F F R 165 ctgaggagaagcgtgcgctcctctcagaacgtcgccatgatcttctttcgg 507 I H N A G S T F T L T V R K H I N 182 attcacaacgccggcagcaccttcacgctgaccgtcaggaaacacatcaat 558 P F P C N V I S Q S P E G S Y T M 199 ccgttcccctgtaatgtcatctcccagtcaccagagggcagttacacgatg 609 V I P Q Q H R K C S F S I I Y P V 216 gtgatcccgcagcagcacaggaaatgcagcttctccatcatctacccggtg 660 E I D V S E F S L G H F N N F P Q 233 gagatcgacgtctctgagttcagcctcggacacttcaacaactttccccaa 711 R S M P G C A E S G D F V Q L L G 250 aggtccatgcccggttgtgcagaatcaggagatttcgtgcagctgttggga 762 G S G I D T S K L L P I T D L C I 267 ggaagcggtatcgacacgtcgaagctgctgcccatcacggacctctgcatc 813 S L L D P T H M K I G C D N T V V 284 tccttactggaccccacccacatgaagatcggctgcgacaacacggtggtg 864 R M V S S G K F V S R V S F S Y R 301 aggatggtgtccagcgggaagtttgtgagccgagtgtcgttcagctacagg 915 L L D S Q E L Q T I K L N N V E D 318 ctactggacagccaggagctgcagaccatcaaactcaacaacgtggaggat 966 F C F N N 323 ttctgtttcaacaactgaacccgacaggatcctccagtgacacacgcatca 1017 tctgactgcaaacattttttaaattctttgaagagccacagatccacccgg 1068 tcgctccatctgattaggtgaaacgtcttaaatccgaagacgtaaacataa 1119 aagaaaaataagatgaattacgatccacgctttttgttttcgttccatttt 1170 tccatcttatttcagtcgttccgttgtcgctgaataaagctcgatgaagtg 1221 tccttttgtgtttggggaactgctattgttttatttttgtgtatttattaa 1272 gacttactgatgatgttgttatttgttacgctgtatgagttgtggtcaaca 1323 ttcttgcaaagggacgggctaaaaaagttaccttctgtttatgttgctgaa 1374 cgacacgcgatgtgccgattcatttcctgcagcaggtgaccaggaggggac 1425 ggtgaagtgttccatgtaataaatacagtgttttcttaatgcggttcaatt 1476 tgtataaaacctttttgtaactcatcgcatgacaaagcaaaaaaaaaaaaa 1527 aaaa -3’ 1531 !!!!!
35
Figure 4. Martos-Sitcha et al. 885
886
887
Figure 4. Martos-Sitcha et al. Sparus aurata --------------------MRVMERTFREQLFFLLLCASVLKGDCRYIEN--NEISKDE 38 Solea senegalensis -----------------------MSLPLRAQLLLFLISLSSKMGISRYIEDS---ESSEE 34 Cyprinus carpio -----------------------MSGTSRAQLCFLLLSVTALRGHARFLDIQDNEISPEG 37 Danio rerio -----------------------MSATSRAQLCFLLLSVTALRGHARFLDMQDNEISPEG 37 Homo sapiens -----------------------MSPNFKLQCHFILIFLTALRGESRYLELR--EAADYD 35 Mus musculus -----------------------MSPNFKLQCHFILILLTALRGESRYLEVQ--EAAVYD 35 Gallus gallus MPRRLLPAGSEQQVLSAHGGAATMPSAFQLQCHLVLILLAASKGDTRYLEVR--DAGEDE 58 Xenopus laevis -----------------------MTPASRPDWCLILLFLAVLRGESRYIQMR--EAAE-D 34 * : : :.*: : * *::: . Sparus aurata LYSFFNSELKRETTEELMYRRPLRCLDMVAVEGQFTFTAERPQLSCAAFFMAEPNEVITV 98 Solea senegalensis LDSLFGVDQK--IKEDFIYRRPLRCLDMLATDGAFTFVASQPQLACAAFIIAEPTQVISV 92 Cyprinus carpio LLSLLSSELKRELPEEFVYRRALRCLDMVAIEGQFTFTAERPQLNCAVFFIGEPSDIISI 97 Danio rerio LLSLLSSELKRELPEEFVYRRALRCLDMVAVEGQFTFTAERPQLNCAVFFIGEPTDVISI 97 Homo sapiens PFLLFSANLKRELAGEQPYRRALRCLDMLSLQGQFTFTADRPQLHCAAFFISEPEEFITI 95 Mus musculus PLLLFSANLKRDLAEEQPYRRALRCLDMLSLPGQFTFTADRPQLHCAAFFIGEPEEFITI 95 Gallus gallus PFLLLSEDLKRELSAGHIYRRSLRCIDMLSIEGQFTFTADQPQLHCATFFIGEPEELLTI 118 Xenopus laevis ALFLLNSDFKRELSEGQIYRRSLRCIDMLSIEGQFTFQADRPQLHCALFLIGEPEEFIII 94 ::. : * ***.***:**:: * *** *.:*** ** *::.** :.: : Sparus aurata EYDNVDIDCRGGDFITVFDGWVMKGEKFPSSQDHPLPLYERYVDYCDSGALRRSVRSSQN 158 Solea senegalensis ELSDVNIDCSAGDFIKMFDGWVLKGEKFPNSQDHQLPLHQRYTDYCSNPATGATSRSSQN 152 Cyprinus carpio EYDSVNIDCRGGDFIKVFDGWVMKGEKFPSTQDHPLPLYKRYSDYCETGVTRPIVRSSQN 157 Danio rerio EYDSVNIDCRGGDFIKVFDGWVMKGEKFPSSQDHPLPLYERYTDYCETGVSRPIVRSSQN 157 Homo sapiens HYDQVSIDCQGGDFLKVFDGWILKGEKFPSSQDHPLPSAERYIDFCESGLSRRSIRSSQN 155 Mus musculus HYDLVSIDCQGGDFLKVFDGWILKGEKFPSSQDHPLPTMKRYTDFCESGLTRRSIRSSQN 155 Gallus gallus EYDFVNIDCQGGDFLKVFDGWILKGEKFPSSLDHPLPTSQRYTDFCESGAVQRSIRSSQN 178 Xenopus laevis EYNFVNIDCIGGDILKVFDGWIIKGEKFPSSLDHPLSTMERYTDICEDGDVGSITRSSQN 154 . . *.*** .**::.:****::******.: ** *. :** * *. ***** Sparus aurata VAMIFFRIHNAGSTFTLTVRKHINPFPCNVISQSPEGSYTMVIPQQHRKCSFSIIYPVEI 218 Solea senegalensis VAMVFFRIHSPGSAFTLVVKKIHNPFPCNVMSQRPEGSFTMVLPHQRRNCSFSIIYPVEI 212 Cyprinus carpio VAMLFFRLHQSGSSFTVTFRKLINPFPCNVVSQTPEGSFTMIIPQQHRNCSFSIIYPVEI 217 Danio rerio VAMLFFRLHQSGSSFTVTFRKLINPFPCNVVSQTPEGSFTMIIPQQHRNCSFSIIYPVEI 217 Homo sapiens VAMIFFRVHEPGNGFTLTIKTDPNLFPCNVISQTPNGKFTLVVPHQHRNCSFSIIYPVVI 215 Mus musculus VAMVFFRVHEPGNGFTITIKTDPNLFPCNVISQTPSGRFTLVVPYQHQNCSFSIIYPVAI 215 Gallus gallus VAMIFFRIHQPGNGFTITVKKSANLFPCNVISQTPSGRFTMVIPHQHRNCSFSIIYPVVI 238 Xenopus laevis VAMIFFRVQQPGHGFTLTIRKIPNLFPCNVISQSMNGRFTMITPHQHRNCSFSIIYPVVI 214 ***:***::..* **:..:. * *****:** .* :*:: * *:::********* * Sparus aurata DVSEFSLG--HFNNFP--QRSMPGCAESGDFVQLLGGSGIDTSKLLPITDLCISLLDPTH 274 Solea senegalensis KLTELSLGQAKSNELLPQRQVWSGCSGSGDYVELLGGNGIDTSKMFPVADLCFSLRGLAQ 272 Cyprinus carpio QIGELSLG--QHNDL---KRSILGCAGSGDFVELLGGNGMDTSKMYPMADLCYSFNGPAQ 272 Danio rerio QIGELSLG--QHNDL---KRSILGCAGSGDFVELLGGNGMDTSKMFPMADLCYSFNGPAQ 272 Homo sapiens KISDLTLG--HVNGLQLKKSS-AGCEGIGDFVELLGGTGLDPSKMTPLADLCYPFHGPAQ 272 Mus musculus KISDLTLG--HLHGLQLKKPA-AGCGGTGDFVELLGGTGLDPSKMMPLADLCYPFLGPAQ 272 Gallus gallus KISDLILG--HLNGLFLKNPS-VGCAGVGDFVELLGGTGLDPSKMFPLADLCHSFHGSAQ 295 Xenopus laevis KIFDLTLG--HFNELQLKKPPPKGCGDAGDFVELLGGAGLDPSKMFPLADLCHSFHGSAQ 272 .: :: ** : : : . ** **:*:**** *:*.**: *::*** .: . :: Sparus aurata MKIGCDNTVVRMVSSGKFVSRVSFSYRLLDSQELQTIKLNNVEDFCFNN- 323 Solea senegalensis MKVGCDNSVVRLVSSGNYINRVSFQYRLLGRNELPKNRENSLENFCSLE- 321 Cyprinus carpio MKVGCDNTVIRMVSSGKFVNRVSFQYRLLGHQELQQMKGNSVEDVCLRA- 321 Danio rerio MKVGCDNTVVRMVSSGKFVNRVSFQYRLLGHQELQQMKGNSVEDVCLRA- 321 Homo sapiens MKVGCDNTVVRMVSSGKHVNRVTFEYRQLEPYELENPNGNSIGEFCLSGL 322 Mus musculus MKISCDNAVVRMVSSGKHINRVTFEYRQLEPFELETSTGNSIPEYCLSSL 322 Gallus gallus MKIGCDNTVLRMVSSGKHINRVTFEYYQLDLQEIENRKENSIEEFCFPGI 345 Xenopus laevis MKIGCDNTVVRMVSSGNFINRVTFEYNQLD-RQLEKKQGNSVEEACFPSD 321 **:.***:*:*:****:.:.**:*.* * :: *.: : *
36
Figure 5. Martos-Sitcha et al. 888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
37
Figure 6. Martos-Sitcha et al. 919
920
921
922
0 h 4 h 8 h 12 h 24 h0
20
40
60
80 SW→LSWSW→SWSW→HSW
a
b,*
cc,*
bb
c,*
b,*
Time (hours)
Cor
tisol
(ng/
mL)
abaa
aa
38
Figure 7. Martos-Sitcha et al. 923
924
925
0 h 4 h 8 h 12 h 24 h0
1.0
1.5
2.0 SW→LSWSW→SWSW→HSW
A
a
b b,*
ab,*
Time (hours)
CR
H m
RN
A ex
pres
sion
(a
rbitr
ary
units
rela
tive
to β-
actin
)
b
a aaa
a
b,*
ab,*
a
0 h 4 h 8 h 12 h 24 h0.0
0.5
1.0
1.5
2.0 SW→LSWSW→SWSW→HSW
B
aab,*
b
c
b,&
c
b
c
c c
ab
c
ab,*
Time (hours)
CR
H-B
P m
RN
A ex
pres
sion
(a
rbitr
ary
units
rela
tive
to β-
actin
)
39
Figure 8. Martos-Sitcha et al. 926
927
928
929
0 7 14 210
50
100
150
200
ControlStarvedRe-fed
a
ab ab
b,*
&
Time (days)
Cor
tisol
(ng/
mL)
a a a
40
Figure 9. Martos-Sitcha et al. 930
931
0 7 14 210.0
0.3
0.6
0.9
1.2
1.5
ControlStarvedRe-fed
A
Time (days)
CR
H m
RN
A ex
pres
sion
(a
rbitr
ary
units
rela
tive
to β-
actin
)
a
a
a a a aaa
0 7 14 210.0
0.5
1.0
1.5
2.0
2.5
ControlStarvedRe-fed
a
b,*
B
Time (days)
CR
H-B
P m
RN
A ex
pres
sion
(a
rbitr
ary
units
rela
tive
to β-
actin
)
a
a
a a
aa